The goal of this proposal is to identify the mechanisms underlying tumor exosome-mediated suppression of NK cell activity. Our preliminary data indicate that pretreatment of BALB/c mice with exosomes produced by TS/A breast tumor cells results in more rapid tumor growth and earlier metastasis. We have identified a novel breast tumor exosomal protein (Jak3BP) that is capable of binding to Jak3 in the NK cells, resulting in the ubiquitination of Jak3. siRNA Jak3BP knockout results indicated, however, that although Jak3BP is required, it is not sufficient for ubiquitination of Jak3. We have now identified two exosomal proteins that interact with Jak3BP, Nedd4 and ubiquilin, which have the potential to enhance the ubiquitination of Jak3 or protect Jak3BP against its degradation in IL-2-stimulated NK cells. Our aims are (1) To use siRNA technology to determine if knockout of exosomal Jak3BP in tumor cells is sufficient for reversal of the inhibition of NK-cell activation mediated by TS/A exosomes, or whether other exosomal proteins, such as Nedd4 and ubiquilin, are required. (2) To use siRNA technology to determine if other exosomal proteins that interact with Jak3BP participate in the ubiquitination of Jak3 in NK cells and to identify the molecular determinants of these interaction. We will determine if modification of Jak3BP regulates its choice of interaction partners and, in parallel, use bioluminescence resonance energy transfer assay technology to identify a peptide that can block the interaction of Jak3 and Jak3BP thus reversing the tumor exosome-mediated inhibition of NK-cell activation. (3) To determine which tumor exosomal proteins contribute to the stabilization of Jak3BP in IL-2 stimulated NK cells, and further determine if the elimination of these exosome proteins causes degradation of Jak3BP thus resulting in the attenuation of inhibition of NK cell activation and prevention of tumor growth. (4) To determine if human breast tumor Jak3BP also regulates the ubiquitination of Jak3 of NK cells, and to determine if Jak3BP is over expressed in patients with ductal adenocarcinoma of the breast. The data generated should elucidate the cellular and molecular basis underlying tumor immunosuppression mediated by the exosomes produced by tumor cells and indicate the feasibility of various therapeutic strategies.